bioprocessing for sustainable production of coloured textiles · another acid red-brown biodye...

Work Package 3 – Synthesis of bio-dyes

Lead contractor for this deliverable: UNISI Coordinator organisation: University of Siena Coordinator: Rebecca Pogni Dissemination Level: CO Confidential, only for members of the consortium (including the Commission Services)

Bioprocessing for Sustainable Production of Coloured textiles

CIP Eco-innovation – First Application and market replication projects ECO/09/256112/SI2.567273

www.biscol.unisi.it

Starting date: September 1st, 2010 Duration: 36 months

Deliverable 3.2 Report including the process parameters for the industrial

production of bio-dyes

Bioprocessing for Sustainable Production of COLoured textile

WP3/Deliverable 3.2 di 26

1. INTRODUCTION ........................................................................................................................................................ 3

2. BIOREACTOR DESIGN............................................................................................................................................. 3

3. BIOSYNTHETIC PATHWAY TO PRODUCE DYE AT PILOT SCALE............................................................. 5

3.1. REVERSE OSMOSIS APPARATUS FOR DYE SOLUTION CONCENTRATION......................................... 6

4. APPENDIX.................................................................................................................................................................. 11

4.1. IDENTIFICATION OF THE DYE STRUCTURE .............................................................................................. 11

4.1.1. SIAR1DYE ............................................................................................................................................................ 11

4.1.2. SIAB1 DYE ........................................................................................................................................................... 16

4.1.3. SIAY1 DYE ........................................................................................................................................................... 19

4.1.4. SIARB1 DYE......................................................................................................................................................... 21

5. REFERENCE LIST.................................................................................................................................................... 26



1. Introduction The synthetic routes that will be used at the semi-industrial scale at SETAS premises have been previously defined at small scale as it is shown in Del. 3.1. Starting from the results achieved in the European project SOPHIED, in which 3 partners of BISCOL Consortium were involved, the development of an original process to create azo bonds and phenoxazine moiety through fungal enzymatic coupling opened the way to new safe and environmental friendly routes to bio-dyes synthesis. Two patents on bio-dyes, with azoanthraquinonic and phenoxazine structures, were deposited (US61078,670; US61078,675) and a bioreactor able to synthesized dyes at pilot scale was created. Starting from the results achieved within the FPVI project SOPHIED, the synthetic pathway (such as optimal range of operational conditions) have been implemented at large scale for the production of bio-dyes in order to achieve good performances. A bioreactor for pre-industrial production for biodyes has been set up and some critical points have been addressed in the present project:

a) the engineering and the control of the bioreactor b) optimization of the downstream processing and improvement of the final dye concentration

through the use of a system based on reverse osmosis. 2. Bioreactor design The experimental setup includes the bioreactor and a computer loaded with LabVIEW and a PC board. The sensors provide data concerning the oxygen, pH and Temperature feedback measurement (the system has been designed to be updated with sensors for pressure, air flow rate and Uv-vis spectrophotometer measurements) required for the PID control system (Figure 1). Sensors measure the target parameters from the pilot, and they are in turn electrically connected to the host computer via the Lab-PC-1200 data acquisition board. The program monitors the parameter measurement, converts them and makes appropriate changes to the control systems via a GPIB card connected to the two DC power supplies. The architecture of the pilot control system is a classic digital system that samples the input signal at discrete time intervals and adjusts the output according to an appropriate control algorithm; LabVIEW enables us to easily and rapidly implement and test alternative control algorithms. Figure 2 shows the path of data through the system.



Figure 1 – Bioreactor design.

Figure 2 - Simplified digital control system diagram. The main characteristics of the pre-industrial reactor are listed in the table below (Table 1).



Table 1 – Characteristic of the bio-pilot.

Parameter Value Total footprint 2.0 x 0.8 m2

Number of Cartridge 4 Volume of the cartridge 2 L/each Total amount of immobilised enzyme 1 L per cartridge Expected flow rate Up to 3 m3/h Electrical consumption 0.5 kW

Laccase from Coriolopsis Polyzona has been chosen as biocatalyst by the immobilization onto glass beads (0.4 to 1 mm) (Figure 3). This type of laccase belongs to the family of Polyporaceae. The main characteristics of the biocatalyst are the following: activity 5 ULac/g, storage up to 4 years at 4°C and product half time life in operation up to 1 month.

Figure 3 – Immobilized laccase from Coriolopsis Polyzona onto glass beads. 3. Biosynthetic pathway to produce dye at pilot scale Different chemical precursors have been tested for the coupling reaction with laccase and those which have given more promising results have been reported in Del. 3.1. Between them a tri-colour set (red (SIAR1), blue (SIAB1) and yellow (SIAY1)) acid biodyes plus another acid red-brown biodye (SIARB1) have been selected for their tinctorial properties. The red and blue ones have then been selected for the scale up.

1) Acid blue dye (SIAB1): 3-methyl-2-benzothiazolinone hydrazone and 4-amino-5-hydroxynaphtalene-2,7-disulfonic acid

2) Acid red dye (SIAR1): 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and 4-amino-1-naphthalensulfonic acid

3) Acid yellow dye (SIAY1): 4-nitro-1-naphthylamine and 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid

4) Acid red-brown dye (SIARB1):

2,5 –diaminobenzene sulphonic acid



The synthesis of bio-dyes has been performed in sodium acetate buffer at pH 5 (total volume 25 kg of water) and at the temperature of 35°C. The pH was fixed at 5 and regulated by the use of sulphoric acid. The precursors initial concentration has been defined at 5 g/L. The ratio between dye precursors has been defined 1:1 except in the case of reaction of naphthalensulfonic reactive with laccase mediators ABTS (ratio 20:1). The precursor tank was equipped with an air inlet (10 liter O2/hr). Four cartridges were placed in the system to maximize the contact time between the solution and immobilized laccase. These cartridges have been chosen for their ease of replacement facility. All cartridges can accept 2 L of immobilized laccase. The synthesis has been monitored by UV-visible spectrophotomer of the bio-pilot in order to prove the formation of the new chemical species, the dyes, from the coupling reactions between precursors. Integration of the whole process was made by a computer, placed next to the trailer. Data from all sensors (pH, Temperature, Oxygen saturation, Color in APHA Units, Pressure,) and actives elements (Mediator injection, status of the pump, injection of new effluent) are logged. The software developed by Wetlands allows the monitoring of all parameters with simple interface. 3.1. Reverse osmosis apparatus for dye solution concentration As the dye synthesis is performed in aqueous solution with a starting precursor concentration of 5 g/L, the main problem to be overcome in the downstream process of dye synthesis, is the reduction of water to obtain a concentration for liquid dye comparable to conventional industrial liquid dye (20-30% w/w). A reverse osmosis process have been selected to obtain a proper concentration of the produced liquid dyes (Table 2). The reverse osmosis apparatus is present in SETAS and it is used to concentrate conventional liquid dyes to sell in the market. An example of this apparatus is shown in Fig. 4. Briefly, the reverse osmosis process works by using a high pressure pump to increase the pressure on the dye solution and force the water across the semi-permeable membrane, leaving around 95-99% of dye behind the reject stream. The amount of pressure required depends on the dye concentration of the feed solution: a more concentrated dye solution required more pressure to overcome the osmotic pressure.

Figure 4 - Image of a reverse osmosis apparatus used in SETAS.



Table 2 – Details of the biosynthetic production of the tri-colour set selected for woollen fabrics.

Chemical structure of the bio-dyes Dye Identification

Name Reaction time (h)

Yield (%)

Final concentrationafter reverse osmosis (g/L)

SIAB1 24 70 47

SIAR1 16 78 15

SIAY1 16 70 20

N N

SO3H

HO3S

NH2H2N

SIARB1 12 70 20

Technical data sheet for liquid biodyes

Dye: SIAR1 Colour index name: Acid Red Precursor Composition: 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and 4-amino-1-naphthalenesulfonic acid (molar ratio 1:1)

SIAR1 dye

CIP-EIP-Eco-Innovation ECO/09/256112/SI2.567273



Physical data: Appearance: liquid Color: red Odor: none Solubility in water: soluble pH: 5 Health effect data: Eye Contact: Wash immediately with large amounts of water, occasionally lifting the upper and lower lids until no evidence of product remain. Skin Contact: Wash immediately with soap and plenty of water. If a temporary skin reaction occurs, it should be treated as allergic contact dermatitis. Ingestion: If swallowed, do not induce vomiting. Qualified medical personnel should remove the product by gastric lavage and catharsis. Get medical attention. Inhalation: If inhaled, remove to fresh air. If not breathing, give artificial respiration, preferably mouth to mouth. If breathing is difficult, give oxygen. Call a physician. Eco-toxicity effect data: EC50 (%) 48 h – UNI EN ISO 6341:1999 : 49 (toxicity rating: minor acutely toxic) Ames mutagenicity test (TA98 and TA100 Salmonella typhimurium strains): no genotoxic effects IC50 (g/L) by Neutral Red Uptake Assay: 0.2 Employee Protection Recommendations: Eye Protection: Goggles Skin Protection: This material may be absorbed via the dermal route if prolonged or widespread skin contact occurs. Employees should avoid skin contact by wearing protective clothing. Long sleeve shirts, pants, rubber gloves and boots are recommended. Additional protection, such as impervious suits, are recommended when potential for dermal contact is significant. Employees should wash their hands and face before eating and drinking and shower thoroughly before leaving work. Respiratory Protection: None Ventilation: Local Other: Emergency showers and eye wash stations should be available. Reactivity Data: Stability: Stable Chemical structure: phenazine Incompatibility (materials to avoid): None known Hazardous Decomposition Products: Like any other organic product combustion will produce carbon dioxide and may produce carbon monoxide. Special Precautions and Storage Data: Handling and Storing Precautions: Reseal container after use. Avoid unnecessary contact.

Dye: SIAB1

SIAB1 dye




Colour index name: Acid Blue Precursor Composition: 3-Methyl-2-benzothiazolinone hydrazone hydrochloride hydrate and 4-Amino-5-hydroxynaphthalene-2,7-disulfonic acid monosodium salt hydrate (molar ratio 1:1)

Physical data: Appearance: liquid Color: blue/violet Odor: none Solubility in water: soluble pH: 5 Human Health effect data: Eye Contact: Wash immediately with large amounts of water, occasionally lifting the upper and lower lids until no evidence of product remain. Skin Contact: Wash immediately with soap and plenty of water. If a temporary skin reaction occurs, it should be treated as allergic contact dermatitis. Ingestion: If swallowed, do not induce vomiting. Qualified medical personnel should remove the product by gastric lavage and catharsis. Get medical attention. Inhalation: If inhaled, remove to fresh air. If not breathing, give artificial respiration, preferably mouth to mouth. If breathing is difficult, give oxygen. Call a physician. Eco-toxicity effect data: EC50 (%) 48 h – UNI EN ISO 6341:1999 : ∼10 (toxicity rating: moderately/minor acutely toxic) Ames mutagenicity test (TA98 and TA100 Salmonella typhimurium strains): no genotoxic effects IC50 (g/L) by Neutral Red Uptake Assay: 0.2 Employee Protection Recommendations: Eye Protection: Goggles Skin Protection: This material may be absorbed via the dermal route if prolonged or widespread skin contact occurs. Employees should avoid skin contact by wearing protective clothing. Long sleeve shirts, pants, rubber gloves and boots are recommended. Additional protection, such as impervious suits, are recommended when potential for dermal contact is significant. Employees should wash their hands and face before eating and drinking and shower thoroughly before leaving work. Respiratory Protection: None Ventilation: Local Other: Emergency showers and eye wash stations should be available. Reactivity Data: Stability: Stable Chemical Structure: azo dye Incompatibility (materials to avoid): None known Hazardous Decomposition Products: Like any other organic product combustion will produce carbon dioxide and may produce carbon monoxide. Special Precautions and Storage Data: Handling and Storing Precautions: Reseal container after use. Avoid unnecessary contact.



Dye: SIAY1 Colour index name: Acid Yellow Precursor Composition: 4-nitro-1-naphthylamine and 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid (molar ratio 1:1) Physical data: Appearance: liquid Color: yellow Odor: none Solubility in water: soluble pH: 5 Health effect data: Eye Contact: Wash immediately with large amounts of water, occasionally lifting the upper and lower lids until no evidence of product remain. Skin Contact: Wash immediately with soap and plenty of water. If a temporary skin reaction occurs, it should be treated as allergic contact dermatitis. Ingestion: If swallowed, do not induce vomiting. Qualified medical personnel should remove the product by gastric lavage and catharsis. Get medical attention. Inhalation: If inhaled, remove to fresh air. If not breathing, give artificial respiration, preferably mouth to mouth. If breathing is difficult, give oxygen. Call a physician. Eco-toxicity effect data: EC50 (%) 48 h– UNI EN ISO 6341:1999 : ∼2 (toxicity rating: moderately acutely toxic) Ames mutagenicity test (TA98 and TA100 Salmonella typhimurium strains): no genotoxic effects IC50 (g/L) by Neutral Red Uptake Assay: 0.4 Employee Protection Recommendations: Eye Protection: Goggles Skin Protection: This material may be absorbed via the dermal route if prolonged or widespread skin contact occurs. Employees should avoid skin contact by wearing protective clothing. Long sleeve shirts, pants, rubber gloves and boots are recommended. Additional protection, such as impervious suits, are recommended when potential for dermal contact is significant. Employees should wash their hands and face before eating and drinking and shower thoroughly before leaving work. Respiratory Protection: None Ventilation: Local Other: Emergency showers and eye wash stations should be available. Reactivity Data: Stability: Stable Chemical Structure: Azo dye Incompatibility (materials to avoid): None known Hazardous Decomposition Products: Like any other organic product combustion will produce carbon

SIAY1 dye




dioxide and may produce carbon monoxide. Special Precautions and Storage Data Handling and Storing Precautions: Reseal container after use. Avoid unnecessary contact. 4. Appendix 4.1. Identification of the dye structure Once synthesized, the chemical identification of the bio-dyes structure has been done by the nuclear magnetic resonance (NMR), high pressure liquid chromatography mass spectra (HPLC-MS), infrared spectra (IR). All the analytical experiments have been performed by the project partners WET, SETAS and UNISI. Toxicity tests on the bio-dyes have been reported in Del. 6.1 and the dyeing properties are reported in Del. 5.3. 4.1.1. SIAR1 dye The dye SIAR1 has been synthetized from the precursors 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and 4-amino-1-naphthalensulfonic acid (molar ratio 1:20). Infrared spectra have been obtained for the red dye and each precursors (Figs. 5-7). The shoulder band at around 3300 cm−1 is due to the N–H stretching vibrations of the –NH group (Fig. 6) [1]. The peak at 3365 cm−1 and the shoulder band at around 3100 cm−1 are ascribed to the asymmetrical and symmetrical of N–H stretching vibrations of NH2 group, respectively. Two strong peaks at 1635 cm−1 and 1533 cm−1 are associated with the stretching vibrations of C=C and C=N group in phenazine ring. The peaks at 1365 cm−1 is associated with C–N–C stretching in the benzenoid. The bands at 764 cm−1 and 687 cm−1, which are the characteristic of C–H out-of-plane bending vibrations of benzene nuclei in the phenazine skeleton, are also observed. Furthermore, the band at around 1100-1200 cm-1 is associated to the sulfonic group from the naphthalene unit of the precursor. From the comparison of the IR spectra for dye and precursors (Figure 8), seems that the chemical structure of the dye is similar to the 4-amino-1-naphthalensulfonic acid and 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) is no part of the structure.



Figure 5 – IR spectra of 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid).

Figure 6 – IR spectra of 4-amino-1-naphthalensulfonic acid.



Figure 7 – IR spectra of SIAR1 dye.

Figure 8 – Comparison between IR spectra of SIAR1 dye and its precursors.

0

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100

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200

250

350 850 1350 1850 2350 2850 3350 3850

cm-1

% T

RedABTS4-A-1-N



In order to know the molecular weight of the product of biosynthesis was performed a mass spectrum, which revealed the presence of a very intense peak at 378 m/z. In the mass spectra also appeared a signal relating to the fragmentation of the radical formed by oxidation of ABTS (256 m/z) [2]. The 1H and 13C NMR spectra of the red dye and its precursor (4A-1NS) are reported in Figures 9 and 10. For both molecules, all the proton signals are within the aromatic region of the NMR spectrum (9.0-6.5 ppm) indicating the aromatic moieties as the main functional groups present in the molecular structure of the dye.

4A‐1NS

RED DYE

Figure 9 - 1H NMR spectrum of the precursor (top) and of the investigated dye (bottom). T=298K.

4A‐1NS

RED

Figure 10 - 13C NMR spectrum of the precursor (top) and of the investigated dye (bottom). T=298K. As easily observed from both spectra Figure 9, the spectrum of red dye contains the NMR signals of 4A-1NS as well, which is indeed used in large excess during the chemical synthesis of the dye. In particular the ratio between the proton integrals of the two molecules allowed to roughly estimate a 60% of the precursor still present in solution. The comparison between the spectra reported in Figure 9, together with the analysis of 2D homonuclear and heteronuclear maps, unequivocally allowed to determine the NMR signals (showed in Table 3) belonging to the investigated dye.



The obtained data are consistent with the presence of a unique spin system composed by four aromatic protons (8.68, 8.19, 7.79, 7.71 ppm) and an isolated aromatic proton (7.97 ppm). In addition the 13C NMR spectra indicated the presence of six non magnetically equivalent quaternary carbons. Table 3 - 1H and 13C NMR parameters (chemical shift (ppm) and JHH (Hz)) of the investigated dye.

1H δ (ppm)* 13C δ (ppm) * 1H molteplicity J (Hz)

8.68 128.4 d 8.5

8.19 125.4 d 8.5

7.97 130.7 s

7.79 130.9 dd 8.5

7.71 129.1 m.

147.4

132.7

131.9

131.0

127.0

118.6

*Chemical Shift are referred to TMSP-d4

In order to get more insights on the molecular structure of the dye, diffusion order DOSY experiments were recorded. Such experiments allow to calculate the molecular diffusion coefficients (D m2s-1) and they are widely used to identify mixture components. The obtained results (Figure 11) clearly show the dye has different diffusion features compared to the precursor.

4A‐1NS

RED DYE

D (m2s‐1)

Dye 4.8x10‐10

4A‐1NS 7.0x10‐10

Figure 11 - NMR DOSY spectrum of the investigated dye. The y-axis shows the D values as log scale. On the other hand the D values are shown in the top inset. T=298K.



In particular the D value of the dye is almost twice smaller than those of 4A-1NS indicating a slower molecular tumbling of the dye. Since the two molecules experience identical solution conditions such slowing down of the tumbling can be only explained by considering a larger molecular mass of the dye. 4.1.2. SIAB1 dye The dye SIAB1 has been synthetized from the precursors from 3-methyl-2-benzothiazolinone hydrazone and 4-amino-5-hydroxynaphtalene-2,7-disulfonic acid (molar ratio 1:1). Infrared spectra have been obtained for the blue dye and each precursors (Figs. 12-15). The peak at around 3400 cm−1 is due to the O–H stretching vibrations. The peak at around 3100 cm−1 are ascribed to the asymmetrical and symmetrical of N–H stretching vibrations of NH2 group. Two strong peaks in the range 1400-1472 cm−1 and 1594 cm−1 are associated with the stretching vibrations of C=C and C=N. The bands at 753 cm−1 and 676 cm−1, which are the characteristic of C–H out-of-plane bending vibrations of benzene nuclei, are also observed. Furthermore, the band at around 1100-1200 cm-1 is associated to the sulfonic group from the naphthalene unit of the precursor. From the comparison of the IR spectra for dye and precursors (Figure 15), seems that the chemical structure of the dye is similar to the 4-amino-5-hydroxynaphtalen-2,7-disulfonic acid.

Figure 12 – IR spectra of 3-methyl-2-benzothiazolinone hydrazone.



Figure 13 – IR spectra of 4-amino-5-hydroxynaphtalen-2,7-disulfonic acid.

Figure 14 - IR spectra of SIAB1 dye.



Figure 15 – Comparison between IR spectra of SIAB1 dye and its precursors. In order to know the molecular weight of the product of biosynthesis was performed a mass spectrum, which revealed the presence of a very intense peak at 493 m/z. The 1H spectra of the dye indicate the presence of many different molecules in solution (Figure 16). In particular the aromatic region of the spectrum contains many different signals at diverse relative abundance. The comparison between the spectra of the dye and those of the two precursors allowed to verify their possible presence in solutions. The obtained data (not shown) indicate that only the precursor H-Acid is present in solution at very low amount.

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cm-1

% T

BlueH-acidMBTH



Figure 16 - 1H NMR spectrum of the investigated dye at T=298K. 4.1.3. SIAY1 DYE The dye SIAY1 has been synthetized from the precursors from 4-nitro-1-naphthylamine and 4-amino-5-hydroxynaphtalene-2,7-disulfonic acid (molar ratio 1:1). Infrared spectra have been obtained for the yellow dye and each precursors (Figs. 17-19). The peak at around 3500-3200 cm−1 is due to the O–H stretching vibrations. The peak at around 3300-3400 cm−1 are ascribed to the asymmetrical and symmetrical of N–H stretching vibrations of NH2 group. Two strong peak at 1600 cm−1 is associated with the stretching vibrations of C=C. The bands at around 800 cm−1, which are the characteristic of C–H out-of-plane bending vibrations of benzene nuclei, are also observed. Furthermore, the bands at around 1100-1200 cm-1 are associated to the sulfonic group from the naphthalene unit of the precursor. Any band structures observed between 3150 and 3000 cm-1 are almost exclusively indicative of unsaturation (C=C-H) and/or aromatic rings. The other most important set of bands are the aromatic ring vibrations centered around 1600 and 1500 cm-1, which usually appear as a pair of band structures, often with some splitting. The peaks at around 1500 cm-1 and 1400-1300 cm-1 (N-O stretching) and 3300-3500 cm-1 (N-H stretching) typical of 4-nitro-1-naphthylamine don’t appear in the IR spectra of the yellow dye. From the comparison of the IR spectra for dye and precursors (Figure 19), seems that the chemical structure of the dye is similar to the 4-amino-5-hydroxynaphtalen-2,7-disulfonic acid.



Figure 17 – IR spectra of 4-nitro-1-naphthylamine.

Figure 18 – IR spectra of 4-amino-5-hydroxynaphtalene-2,7-disulfonic acid.



Figure 19 –IR spectra of SIAY1 dye. In order to know the molecular weight of the biosynthesized product a mass spectrum was performed. A very intense peak at 500 m/z was detected. 4.1.4. SIARB1 dye The dye SIARB1 has been synthetized from the precursor 2,5-diaminobenzensulfonic acid. Peroxidase and laccase oxidize aromatic amines and phenols generating radical species which then are coupled to form dimers. The subsequent oxidation and coupling reactions are shown below (Figure 20).

SO3H

NH2

NH2

SO3H

NH

NH2

SO3H

NH

NH2

SO3H

NH2

NH

SO3H

NH2

NH

SO3H

NH2

NH

SO3H

NH2

HNn

Figure 20 – Radical formation and coupling reaction of 2,5-DABS catalyzed by laccase. IR spectra performed on solid samples of the precursor 2,5-DABS and the product is shown in Figure 21. Immediately it is possible to notice that the two spectra are similar. Of great importance for the purposes of our study are the absorption bands centered at values close to 3400-3300 cm-1 which can be assigned to the stretching vibrations of the NH bonds. This experimental relevance



proved that some of the amino groups present in the starting substrate did not undergo oxidative processes by the laccase because are found also in the final product.

Figure 21 – IR spectra performed on solid samples of 2,5-DABS and its product obtained by biosynthesis. The 1H-NMR spectrum gave results similar and complementary to those obtained by IR spectroscopy (Figure 22). Making an analysis of both compounds solubilized in DMSO, a very broadband can be observed and assigned to the chemical shift values of the amino groups. This information can not be obtained from NMR studies with deuterated water due to the fast proton exchange between the solvent and the hydrogens of the amino groups.

pprreeccuurrssoorr pprroodduucctt

PRODUCT

PRECURSOR

A)



Figure 22 –1H 1D NMR spectrum of precursor and product dissolved in DMSO at T = 298K: A) range 9-6.5 ppm and B) range 7.5-6.5. Focusing on the high intensity couplings, it is possible to deduce that the behavior for the two species is the same. The small shift of the peaks in the spectra is attributable to the content of residual H2O in the final product following the process of evaporation of the solvent and which influence the chemical shift of the analyzed species (Figure 22 and Figure 23).

Figure 23 – Chemical structure of the 2,5-diaminobenzensulfonic acid. The singlet is attributable to hydrogen in the 6-position away from the other proton interactions, while the two doublets are associable to H4 and H3 from the spectra of correlation. In order to eliminate the contribution from residual H2O, were carried further analysis of 1H NMR for the precursor and the product dissolved in D2O (Figure 24). The spectra are similar, although in the product peaks tend to be larger for the greater size of the molecule. It is evident, however, that must be present in the product symmetry.

PRODUCT

PRECURSOR

B)

6 43

SO3H

NH2

H2N

1 2

3 4

5

6



Figure 24 – One dimensional 1H NMR spectrum of precursor and product dissolved in DMSO at T = 298K in the range 8.2-7 ppm. Furthermore, 13C NMR analysis has been made (Figure 25a) to detect the different substituents linked to the carbon atoms of the aromatic structures. It was observed that the chemical shift on carbon-2 (C2) of 2,5-DABS is different from that of the product, while all other values are the same. This consideration is seen in Figure 25b. From this analysis it is possible to deduce that the compound obtained by the enzymatic biosynthesis presents the characteristics of symmetry because only the substituents linked to C2 in the product differ from those of the precursor.

Figure 25 –13C 1D NMR spectra of precursor (red) and product (balck) dissolved in DMSO at T = 298K: (a) range 146-116 ppm and (b) range 128-118. The assignment of peaks in the spectra was possible by observing the two-dimensional NMR spectrum of heteronuclear correlation between 1H and 13C (HSQC) that allowed us to observe the pair of 2.5-DABS and also of the product (Figure 26).

PRODUCT

PRECURSOR

5 1

4 6

2

3

6 4 3

2

(a)

(b)



Figure 26 –1H-13C 2D HSQC-NMR spectra of precursor and product dissolved in DMSO at T = 298K.

In order to approximately estimate the size of the product were performed diffusion measurements that allow to identify if there are differences in molecular weight between the precursor and the product. As already mentioned, these analysis gives a direct proportionality between the molecular weight of the compound and the diffusion. The latter, in fact, directly depends on the molecular shape and more precisely from the hydrodynamic radius. The low variation of the diffusion value for the precursor and the product (Table 4), proved that the product is a small molecule; from these values it is expected that the molecular weight of the product does not exceed 4-5 monomeric units, which suggests that does not form a large dimension polymer. Table 4 – Diffusion coefficients for the precursor and the product.

precursor product

H2O 19.0 19.3

Since the IR and NMR spectra shows the presence of -NH2 groups, the symmetrical property of the molecule, the molecule is soluble in water and polar solvents (eg: methanol), but not in other organic solvents, and finally the sulfonic group is maintained. In order to know the molecular weight of the biosynthetic product, a mass spectrum in the negative mode was performed. The presence of a very intense peak at 369 m/z was revealed without the formation of an extensive fragmentation and the creation of multiple adducts. The following molecular structure shown in Figure 27 was assumed.

N N

SO3H

HO3S

NH2H2N

Figure 27 – Dimeric structure for the product obtained by the biosynthesis of 2,5-DABS.



5. Reference list [1] Hao A., Sun B., Yang X., Lu L., Wang X. Synthesis and characterization of poly (o-phenylenediamine) hollow multi-angular microrods by interfacial method. Materials Letters 63 (2009) 334–336. [2] Marjasvaara A., Janis J., Vainiotalo P. Oxidation of a laccase mediator ABTS as studied by ESI-FTIRCR mass spectrometry. Journal of Mass Spectrometry 43 (2008) 470-477. [3] Alva K.S., Kumar J., Marx K.A., Tripathy S.K.. Enzymatic Synthesis and Characterization of a Novel Water-Soluble Polyaniline: Poly(2,5-diaminobenzene sulfonate), Macromolecules 30 (1997) 4024-4029. [4] Enaud E., Trovaslet M., Bruyneel F., Billottet L., Karaaslan R., Sener M.E., Coppens P., Casas A., Jaeger I.J., Hafner C., Onderwater R.C.A., Corbisier A.-M., Marchand-Brynaert J., Vanhulle S. A Novel Azoanthraquinone Dye made through Innovative Enzymatic Process. Dyes and Pigments, 85 (2010) 99-108.

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